Virotherapy with oncolytic viruses (OV) is a new form of therapy for the treatment of malignant tumor diseases, which has demonstrated its effectiveness in several clinical trials in the last two decades (Fukuhara et al., 2016). The anti-tumor effect is based on a dual mechanism. One is the destruction of the tumor cells by the tumor cell-specific replication of the OV, wherein seemingly OVs do selectively replicate in cancer cells, spread within tumor tissue and lead to tumor destruction. On the other hand due to the viral replication immunological processes are induced, which leads to a systemic anti-tumoral immune response (Kaufman et al., review 2015). Various DNA viruses, such as adenovirus, parvovirus, vaccinia virus and herpesvirus, and RNA viruses, such as Coxsackie-A-virus (CVA), vesicular stomatitis virus and reoviruses have been used as OV for treatment of cancer.
RNA viruses have a short replication cycle and produce a very large number of progeny, which gives them an advantage relative to DNA viruses in oncolytic virotherapy. Moreover, RNA viruses may be safer, as they lack the potential for genotoxic effects caused by integration into the host genome. In the course of the last fifteen years, enteroviruses such as CVA21, echovirus 1 and poliovirus, which are single-stranded RNA-viruses and belong to the Picornaviridae family, were evaluated for their potential as oncolytic agents against melanoma, breast and prostate cancer (Berry et al., 2008; Shafren et al., 2004; Skelding et al., 2009). Poliovirus and Coxsackivirus of strains A and B from the genus Enterovirus (EV) of the family Picornaviridae have been considered as promising OV for the therapy of tumor diseases.
Recently, another member of this group, Coxsackievirus B3 group (CVB3), strain Nancy, was described as a novel OV for treatment of lung carcinomas. The most comprehensive studies concerning effectiveness so far have been conducted with the oncolytic poliovirus variant PVS-RIPO in glioblastomas and Coxsackievirus A21 (CVA21) in melanomas. Of the Coxsackie B viruses (CVB), only CVB3 Nancy as OV in lung carcinomas was comprehensively described in an in vivo mouse model (Miyamoto et al., 2012). There it was shown that wild-type CVB3 (Nancy) can lyse human lung carcinoma cell tumors in nude mice. At the same time, the authors showed that although CVB3 (Nancy) induced inflammatory side effects, they were not considered as severe and did not lead to the death of the animals. No treatment-related mortality, but moderate hepatic dysfunction and mild myocarditis were reported to be the main side effects of CVB3 Nancy treatment of lung carcinoma in mice.
A number of CVB3 strains have been characterized by their tissue tropism and organ toxicity in order to better understand virus-host interaction and pathogenesis caused by viral infection. Among these CVB3 strains, there are strains which are highly cardiotropic, such as CVB3 H3, 31-1-31, M2, HA or H310A1, whereas a number of other strains have been found to be low or non-cardiotropic, e.g. Nancy and PD. There are also CVB3 strains that preferentially infect the liver. Moreover, almost all known CVB3 strains are able to infect the pancreas.
The difference in pathogenicity is believed to be attributable to viral capsid proteins, which are directly involved in virus-cell attachment and virus uptake. In general, most of CVB3 strains utilize the Coxsackievirus and adenovirus receptor (CAR) as primary and the decay accelerating factor (DAF) as co-receptors to infect cells (Bergelson et al., 1998; Bergelson et al., 1995). However, recently it was found that the strain CVB3 PD has an additional and unique receptor tropism as it can use heparan sulfates (HS) to enter the cells (Zautner et al., 2003).
Furthermore, as it was previously shown that CVB3 Nancy induces severe inflammation of the pancreas and the heart in mice (Stein et al., 2015; Pinkert et al., 2009) and in addition, that some CVBs are associated with the development of inflammatory and dilated cardiomyopathies in humans (Andreoletti et al., 2009; Andreoletti et al., 2007) or that CVB3 (Nancy) has been described in connection with severe infections of children (Ronellenfitsch et al., 2014), it seemed questionable to what extent CVB3 Nancy could actually be considered a safe OV.
It is therefore an object of the present invention to overcome the described disadvantages of the state of the art OVs in the treatment of cancer, especially with respect to efficiency and safety of used OVs. Moreover, it is the aim of the present invention to provide a new variant of CVB3 with substantially improved oncolytic efficiency and safety compared to known strains such as e.g. CVB3 strain Nancy.